Dominant‐negative inhibition of breast cancer resistance protein as drug efflux pump through the inhibition of S‐S dependent homodimerization

Abstract

Key words: drug resistance; ABC transporter; mitoxantrone; retrovirus vector Tumor cells that acquire resistance to certain chemotherapeutic agents sometimes develop cross-resistance to other structurally unrelated drugs. 1 This phenomenon is known as multi-drug resistance. A family of ATP-binding cassette (ABC) transporters such as MDR1 gene product P-glycoprotein 1-4 and MRP1 5 is involved in multidrug resistance. They pump out various structurally unrelated antitumor agents in an energy-dependent manner.Breast cancer resistance protein (BCRP), also called ABCP or MXR, is a newly discovered ABC transporter, 6 -8 which is a half molecule with a C-terminal transmembrane segment and an Nterminal ATP-binding site. 9 The BCRP gene was first identified as an ABC transporter overexpressed in the placenta. 7 BCRP overexpression is reported in both mitoxantrone-resistant and topotecan-resistant cells. 10 -12 Because resistance to mitoxantrone, SN-38 and topotecan develop concurrently, BCRP presumably acts as an efflux pump, resulting in decreased intracellular concentrations of these anticancer agents. 13,14 Many ABC transporter proteins have two transmembrane segments and two ATP-binding sites. 15 Some half-molecule ABC transporters are known to form homodimers or heterodimers. 16 -18 BCRP is also assumed to act as a functional dimer, however its counterpart has not yet been identified. To determine a protein that forms an ABC complex with BCRP, cells were transfected with HA epitope-or Myc epitope-tagged BCRP cDNAs and cell lysates of the transfectants were used to analyze proteins interacting with BCRP by immunoprecipitation and Western blotting. Our results indicate that BCRP forms an S-S homodimer and therefore it is possible to inhibit the function of BCRP by the introduction of a dominant-negative BCRP mutant. MATERIAL AND METHODS Cell cultures and drug sensitivity assayPA317 amphotropic retrovirus packaging cells were grown in high-glucose Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 . The sensitivity of the cell lines to mitoxantrone and SN-38 was evaluated by the inhibition of cell growth after incubation at 37°C in the presence of various concentrations of the drugs examined. Six days after incubating cells with the drugs, cell numbers were determined by flow cytometry using a Coulter cell counter and drug concentrations that inhibited cell growth by 50% (IC 50 ) were evaluated. 19 Statistical analysis was performed using Student's t-test. Vectors and transfectantsBriefly, complementary DNA for human BCRP was isolated by PCR using Marathone-ready human placenta cDNA library (Clontech, Palo Alto, CA) as a template. Synthetic oligonucleotides corresponding to the Myc epitope peptide (EQKLISEEDL) or HA epitope peptide (YPYDVPDYA) were added upstream from the ATG codon of BCRP cDNA by PCR and termed MycBCRP and HABCRP, respectively. The nucleotide sequences of independently isolated BCRP cDNA clones were analyzed using an automate...

Mutational Studies of G553 in TM5 of ABCG2: A Residue Potentially Involved in Dimerization

“…In the cross-linking experiments presented in this manuscript ( Figure 5 and Figure 9/B) a stronger signal was observed on immunoblot in the samples treated with the cross-linking agents compared to the non-treated ones. This phenomenon has not been explained, although, it has been reported by other investigators and in our previous studies as well (21) (33) As the lower protein expression levels seen on immunoblot and the lack of surface expression might be the result of abnormal folding and/or dimerization, using the baculovirus heterologous expression system, we expressed the G553L mutant in Sf9 insect cells (Spodoptera frugiperda ovarian cells), which might be more tolerant of an aberrant protein and typically yields higher levels of protein expression (34). Interestingly, we found that the G553L mutant migrates to the cell surface in the insect cells as demonstrated by flow cytometry with the 5D3 antibody ( Figure 6A).…”

Novel Acrylonitrile Derivatives, YHO-13177 and YHO-13351, Reverse BCRP/ABCG2-Mediated Drug ResistanceIn VitroandIn Vivo

“…SN-38-resistant cells were cloned using the limiting dilution technique, and one well-growing clone, A549/SN4, was selected for further experiments. HCT116/BCRP and P388/BCRP cells were established by the transduction of HCT116 and P388 cells, respectively, with the HaBCRP retrovirus, a Ha retrovirus carrying Myc-tagged human BCRP cDNA (25). K562/ MDR cells were established by the transduction of K562 cells with the HaMDR retrovirus, a Ha retrovirus containing human MDR1 cDNA (26).…”

Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2)

“…BCRP transport function can be inhibited by dominant-negative BCRP mutants, strongly suggesting that homodimer formation is essential for BCRP function (Kage et al, 2002). BCRP mutants, generated by PCR mutagenesis, were transfected into PA317 cells and tested for the loss of BCRP function.…”